Dihydromyricetin Attenuates Metabolic Syndrome And

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Open Access Full Text Article ORIGINAL RESEARCH Dihydromyricetin Attenuates Metabolic Syndrome And Improves Insulin Sensitivity By Upregulating Insulin Receptor Substrate-1 (Y612) Tyrosine Phosphorylation In db/db Mice

This article was published in the following Dove Press journal: Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy

Jidong He1 Purpose: Dihydromyricetin (DHM), the main bioactive flavonoid in vine tea, exerts multi- Junpeng Zhang1 ple health beneficial effects. This work aimed to identify whether a naturally derived Lijuan Dong1 flavonoid product, DHM, can significantly attenuate metabolic syndrome and improve Xuefeng Dang1 insulin sensitivity. Li Wang2 Methods: 10-week-old db/db mice were randomly assigned to receive the antidiabetic agent metformin (Met, 50 mg/kg BW), DHM (1.0 g and 0.5 g/kg BW) or placebo and were Long Cheng 3 simultaneously fed a high-fat diet for 8 weeks. The general status of the animals was Yunxiang Huang 4 observed and recorded daily, body weight and blood glucose levels were measured weekly, 1Department of Gastroenterology, Baoji during the experimental period. On day 55, the oral glucose tolerance test (OGTT) was People’s Hospital, Baoji, Shanxi 721000, performed. After OGTT, all animals were anesthetized and sacrificed by cervical decapita- People’s Republic of China; 2Department of Diabetic Nephropathy, Baoji Central tion. Blood samples were collected in tubes to detect plasma insulin and the biochemical Hospital, Baoji, Shanxi 721008, People’s parameters of lipid metabolism. Pancreas histological changes and islet fibrosis were demon- Republic of China; 3Institute of Medicinal Plant Development, Chinese Academy of strated by H&E staining and Masson staining, respectively. Moreover, the expression of Medical Sciences & Peking Union Medical insulin receptor substrate-1 and phosphorylated insulin receptor substrate-1 in the insulin ’ College, Beijing 100193, People s signaling pathway was detected by Western blot assay. Republic of China; 4Department of R&D, Asparagus Engineering Research Center Results: The oral administration of DHM (1.0 g and 0.5 g/kg BW) reduced the fasting blood of Hebei Province, Qinhuangdao 066008, glucose, serum insulin, and glycated hemoglobin levels and the insulin resistance (HOMA- ’ People s Republic of China IR) index. Furthermore, DHM intervention decreased body weight and the serum lipid profile. In addition, DHM treatment also markedly decreased the relative abdominal fat weight. Western blot analysis indicated that DHM upregulated the IRS-1 (Y612) tyrosine phosphorylation, improving insulin resistance. Treatment with dihydromyricetin attenuated the progression of insulin resistance and pancreatic fibrosis in fatty db/db mice. Conclusion: In summary, we determined the antimetabolic syndrome effect of DHM in db/db obese mice. DHM upregulates the IRS-1 (Y612) tyrosine phosphorylation, improving insulin resistance. Therefore, DHM is a promising therapeutic candidate for the control of metabolic Correspondence: Li Wang Baoji Central Hospital, No. 8, Jiangtan syndrome. ’ Road, Baoji, Shanxi 721008, People s Keywords: dihydromyricetin, metabolic syndrome, hypoglycemic function, insulin Republic of China Email [email protected] resistance, insulin receptor substrate-1

Long Cheng Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Introduction Peking Union Medical College, No. 151, Malianwa North Road Haidian District, Currently, there is no agreed upon definition of metabolic syndrome (MetS). The Beijing 100094, People’s Republic of China most commonly used definition is the National Cholesterol Education Program Tel/Fax +86 10 57833013 Email [email protected] (NCEP) definition, which defines MetS as a cluster of key cardiovascular risk factors submit your manuscript | www.dovepress.com Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy 2019:12 2237–2249 2237 DovePress © 2019 He et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php – http://doi.org/10.2147/DMSO.S218487 and incorporate the Creative Commons Attribution Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). He et al Dovepress for diabetes and cardiovascular diseases that includes central abdominal obesity, hypertension, dyslipidemia, and hyperglycemia.1–7 A recent report estimated that 20–25% of the adult population is affected by MetS worldwide. At the same time, the Asia-Pacific region has seen a dramatic increase in the prevalence of obesity, type 2 diabetes and cardiovascular disease.8–11 This rapid increase in MetS has been paralleled by the growing epidemic of type 2 diabetes, hypertension, and cardiovascular disease. Powerful and efficient strategies aimed at primary prevention are urgently needed to curb a further increase in the epidemic and to reduce the morbidity and mortality associated with MetS. The intrinsic causes of MetS include abdominal obesity, physical inactivity, genetic factors and aging.1,2 The most widely recognized metabolic risk factors are hypertension, Figure 1 Relative abdominal fat weight and body weight of db/db mice. The relative abdominal fat weights of the model control, positive control, DHM (0.5 and 1.0 g/kg dyslipidemia (high triglycerides and lower HDL), hypergly- BW) groups were 9.79± 2.76, 8.91± 2.83, 8.98± 2.64, 8.20±1.61 g/100 g body cemia and central obesity.4,5 MetS pathophysiology is com- weight, respectively. DHM intervention at the high dose (1.0 g/kg b.w) induced a significant difference from the model control group. ##p<0.05 compared with the plex, with insulin resistance and the abnormal regulation of normal group. **p<0.05 compared with the DM group. lipid metabolism playing key roles. Traditional Chinese medicine has played a key role in the healthcare system in the past.12–14 The screening of candidate compounds from Chinese medicine is a good strategy for new drug development.15,16 Vine tea, a traditional Chinese materia medica, is composed of the tender stem and leaves of Ampelopsis grossedentata (Hand.- Mazz) W. T. Wang. Vine tea therapy, as a folk treatment, has been applied widely in southern China. According to previous literature and traditional Chinese medicine records, vine tea can eliminate heat and dampness, pro- mote diuresis and blood circulation, and remove blood stasis and channel obstructions.17–21 Dihydromyricetin (DHM), the main bioactive flavonoid in vine tea, exerts antioxidant, anti-inflammatory, antibacterial, anti-intoxication, and anticancer effects.21–33 In the recent report, DHM Figure 2 Fasting blood glucose levels of db/db mice from weeks 1 to 8. Fasting blood glucose levels of untreated db/db mice and db/db/mice treated with the oral protects against memory impairment. This work aimed to administration of metformin and DHM (DHM-h, 1.0 g and 0.5 g/kg BW). DHM fi exhibited a time-dependent hypoglycemic effect with sustained administration. con rm the antimetabolic syndrome effect by evaluating Normal: C57BL, DM: untreated metabolically abnormal obese db/db mice, Met: hypoglycemia, the regulation of lipid metabolism and anti- db/db mice treated with metformin, DHM-h, l: db/db mice treated with DHM at 1.0 g and 0.5 g/kg BW. oxidant function in high fat diet-exposed metabolically abnormal obese mice. ® single-use strips (One-Touch Ultra , lot number: 3662321) were purchased from Shanghai Johnson & Johnson Medical Methods Device Co., Ltd. (Shanghai, China). We used 0.5% carboxyl Chemical Reagents methyl cellulose solution as a vehicle in the animal trials. Dihydromyricetin was extracted from vine tea (Ampelopsis Metformin (50 mg/kg BW, suspended in 0.5% carboxyl grossedentata) and was then purified by recrystallization (the methyl cellulose solution, Squib Pharmaceutical Co., Ltd., chemical formula is shown in Supplementary Figure 1). The Shanghai, China) was applied as a positive reference drug for purity was above 95%, as analyzed by HPLC with a UV hypoglycemic activity measurement. Anti-IRS1(phosphor detector (Supplementary Figure 2). Blood glucose meter and Y612) antibody (synthetic peptide corresponding to the

2238 submit your manuscript | www.dovepress.com Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy 2019:12 DovePress Dovepress He et al epitope DGYMP with a single phosphorylation site Tyr 612, antimetabolic syndrome effect of DHM under experimen- from human IRS1). Abcam plc (Cambridge Biomedical tal conditions and administered the treatment orally each Campus, Cambridge, CB2 0AX, UK). The other chemical day for 8 weeks. Then we use five-fold (0.5 g/kg) as the regents were obtained from Merck KGaA, (Darmstadt, low dose correspondingly. Germany). According to previous literature and traditional Chinese medicine records, vine tea (Ampelopsis grosse- Experimental Animal dentata), as Chinese herbal medicine for treating disease, To confirm antimetabolic syndrome activity, high-fat diet- the recommended dose is 15–30 g per subject daily, or exposed db/db mice were used in this trial as metabolically approximately 0.25–0.5 g/kg body weight for adults (aver- abnormal obese animal models. C57BL/KsJ mice (8 weeks age body weight of 60 kg). DHM content in vine tea is old, as normal control animal) were obtained from SLAC approximately 20–30%. As the botanical drug, the equiva- Laboratories Animal Co., Ltd. (Shanghai, China). All ani- lent DHM dose in mice is 0.5–1.5 g/kg. All mice received mal treatments were in accordance with ethical principles the assigned treatment daily. The general status of the and corresponding standards. The experimental mice were animals was observed and recorded daily. The body evaluated and housed under controlled conditions (tem- weight, food intake, and water intake of the mice were perature, 23°± 2° C; relative humidity, 50% ± 10%; 12-h measured weekly and statistical analyzed. light/dark cycle) and allowed free access to the high-fat To detect fasting blood glucose levels (FGB), we drew diet (45% fat, 20% protein and 35% carbohydrate) and micro blood samples from the tail vein of the mice weekly. deionized distilled water. The experimental protocol was After placing fresh blood (approximately 50 μl) on single- reviewed and approved by the Animal Care and Use use test strips, we used a validated One-Touch Basic Committee of the Animal Resource Center, Institute of Glucose Monitoring Set to detect the glucose content. Basic Medicine, Chinese Academy of Medical Sciences The oral glucose tolerance test (OGTT) was performed (License No: SCXK 2016–0021). The animal welfare fol- on day 55. All animals were fasted overnight, FBG was lowed in this study was guideline for ethical review of detected and then the animals were given a dose of 20% animal welfare in the People’s Republic of China. glucose solution (3.0 g/kg BW). The plasma glucose levels were detected 30, 60, 120, and 180 min after the ingestion Experimental Protocol of a single high dose of glucose to calculate the area under Fasting blood glucose was determined after 2 weeks of the glucose response curve (AUC, min·mmol/L). After 8 adaptive feeding. The hyperglycemic mice (db/db) were weeks of antidiabetic drug or DHM intervention, the mice randomly divided into five groups, each containing ten were fasted for 5 h and anesthetized with diethyl ether. mice. The C57BL mice (normal control group) and the Then, blood samples were collected for biochemical ana- control metabolically abnormal obese db/db model mice lysis. The liver tissue was harvested and prepared for (the placebo-treated model control group) received an tissue protein extraction and Western blot analysis. equal volume of 0.5% carboxyl methyl cellulose solution. Biochemical analysis: After fresh blood was collected, The positive control group (model + Met group) received the blood samples were centrifuged at 3000 x g for 15 min, the antidiabetic agent metformin (Met, 50 mg/kg BW, an separated, removed and stored in an ultra-refrigerator equivalent dose). The DHM-treated mice (model + DHM (−60°C) for further plasma analysis. The plasma insulin groups) received DHM at a dose of 1.0 g and 0.5 g/kg BW. levels were detected by radioimmunoassay immediately. The 10-week-old db/db mice were fed a high-fat diet (45% We used commercial assay kits to estimate the plasma fat, 20% protein and 35% carbohydrate) during an 8-week HbA1c level and the plasma lipid profile according to a experimental period. As a functional food, the recom- standard validated assay. mended dose of vine tea is 3 g per subject daily, or The homeostasis model assessment-estimated insulin approximately 0.05 g/kg body weight for adults (average resistance index has been widely used to estimate insulin body weight of 60 kg). DHM is the most abundant com- resistance in humans with high sensitivity and specificity.34 ponent of vine tea, and the DHM content in vine tea is Here, the HOMA-IR index was used to estimate the change approximately 20–30%. The equivalent DHM dose in in insulin resistance in metabolically abnormal obese db/db mice is 0.1–0.15 g/kg. Here, we used a ten-fold higher mice treated with DHM. The HOMA-IR index was calcu- dose (1.0 g/kg) in mice as the high dose to evaluate the lated by multiplying fasting plasma insulin levels by fasting

Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy 2019:12 submit your manuscript | www.dovepress.com 2239 DovePress He et al Dovepress plasma glucose levels and then dividing by the constant eosin (HE). The pathological deterioration of the aorta 22.5, as shown in the following formula: inner wall was detected with an optical microscope HOMA-IR=fasting blood glucose (mmol/l) × insulin (Leica Application Suite) and recorded. (μU/mL)/22.534,35 Pancreatic tissue sections were fixed in formalin, stained with hematoxylin-eosin (HE), and observed under a light Protein Extraction And Western Blot microscope using the Leica Application Suite. To prepare Analysis the harvested tissue for examination by light microscopy, we removed pancreatic sections, preserved them in 10% To analyze the expression of proteins involved in insulin neutral phosphate-buffered formalin and processed them by resistance, liver tissue was harvested, cut and homogenized routine paraffin sectioning and staining with hematoxylin- on ice in RIPA buffer solution [50 mM Tris (pH 7.6), 150 eosin (HE). Masson staining was performed to detect and mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, analyze islet fibrosis. Staining was performed according to 0.1% sodium dodecyl sulfate (SDS), and 2 mM EDTA] the manufacturers’ instructions. We observed pathological containing protease inhibitor and phosphatase inhibitor. changes using an optical microscope. Then, the supernatants were separated and collected after centrifugation at 12,000 x g for 25 min at 4°C. A bicinch- oninic acid assay was used to measure the protein concen- Statistical Analysis tration, and the protein was stored at −80°C for further Data were recorded and collected properly, then statisti- Western blot analysis. cally analyzed with SPSS software package (Version 19.0, The above protein fractions were separated by 10% IBM Inc., Armonk, New York, USA). All values are sodium dodecyl sulfate polyacrylamide gel electrophoresis expressed as mean values and standard deviations. The (SDS-PAGE) and then transferred onto nitrocellulose quantitative data were normally distributed (as determined membranes in Tris-glycine buffer at 110 V for 1 h. The by the Kolmogorov and Smirnov test). For HOMA-IR, membranes were blocked with 5% (w/v) skimmed milk FBG, HbA1c, lipids, and AUC data, comparisons between powder in Tris-buffered saline containing 0.1% (v/v) groups were analyzed by one-way ANOVA, and group Tween-20 (TBST) and then incubated overnight with comparisons were analyzed using the Student-Newman- appropriate primary antibodies at 4°C. Afterwards, they Keuls test. P values <0.05 were regarded as statistical fi were washed twice with TBST and incubated with second- signi cance. ary antibodies for 2 h at 20–30°C. The results were visua- lized by enhanced chemiluminescence. The relative band Results intensity was determined using computerized densito- Decreased Body Weight And Relative metric analysis. Abdominal Fat Weight The intake of a high-fat diet for 8 weeks led to a further body Histopathologic Examination weight increase in metabolically abnormal obese db/db mice, After collecting blood samples, we quickly removed the and the final body weight of the model control was 69.6 ± 3.5 g. aorta between the aortic valve cusps and the iliac bifurca- At the eighth week, the final body weights were 69.6 ± 3.5 g, tion and stored the samples in 4% paraformaldehyde for 65.2 ± 3.7 g, 67.4 ± 3.6 g and 65.7 ± 4.4 g for the model control, histological examination. Next, we cut consecutive cross- positive control, and DHM (0.5 and 1.0 g/kg BW) groups, sections of the aorta and stained them with hematoxylin- respectively (Table 1).

Table 1 Effect Of DHM On Body Weight In High-Fat Diet Induced db/db Mice

Treatment Dose(g/kg) 0 Week 1 Week 2 Week 3 Week 4 Week 5 week 6 Week 7 Week 8 Week

Control – 23.2±1.3 24.6±2.4 26.0±4.3 29.3±2.1 31.1±2.3 32.6±3.2 33.2±3.1 35.1±3.2 36.0±4.2 DM – 50.7±3.3* 52.8±5.1 56.7±3.5 58.9±3.3 60.2±2.7 61.6±4.3 63.4±3.2 67.8±2.7 69.6±3.5 † † † † † Met 0.05 50.8±3.2 52.8±3.9 53.6±3.8 54.6±3.8 56.9±2.6 59.0±4.5 59.6±3.6 62.7±2.9 65.2±3.7 † DHM 0.5 51.0±4.4 52.1±4.8 54.4±3.9 55.1±4.3 57.9±4.5 58.8±4.7 60.7±4.0 63.8±4.0 67.4±3.6 † † † † DHM 1.0 50.5±3.5 52.1±3.2 54.7±3.1 54.4±3.0 57.8±4.3 58.9±4.4 59.2±3.8 62.4±4.6 65.7±4.4

† Notes: Different from control group: *P < 0.05, Different from model control group: P < 0.05. Number of mice= 10.

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There was no significant difference in food or water con- sumption among the four groups (data not shown). Db/db mice were extremely obese and inactive. There was no difference in physical activity among the four groups. As Figure 1 shows, the relative abdominal fat weights for the model control, positive control, and DHM (0.5 and 1.0 g/kg BW) groups were 9.79± 2.76, 8.91± 2.83, 8.98± 2.64, 8.20 ±1.61 g/100 g body weight, respectively. Intervention with a high dose (1.0 g/kg BW) of DHM reduced the abdominal fat weight, with a significant difference compared to the model control group. As Table 2 shows, the liver weight for the model control, positive control, and DHM (0.5 and 1.0 g/kg BW) groups were 2.99±0.24, 2.62±0.35, 2.65±0.31, 2.61± 0.33 g, respectively. Intervention with a high dose (1.0 g/kg BW) of DHM reduced the liver weight and liver weight/body weight, Figure 3 HbA1c levels in db/db mice at week 8. DHM treatment significantly reduced the HbA1c level. Normal: C57BL, DM: untreated metabolically abnormal with a significant difference compared to the model control obese db/db untreated mice, Met: db/db mice treated with metformin, DHM-h, l: db/db mice treated with DHM at 1.0 and 0.5 g/kg BW. ##p<0.05 compared with the group. normal group. **p<0.05 compared with the DM group.

Decreased Fasting Blood Glucose Levels groups was decreased significantly compared to that in the And HbA1c Levels And Improved diabetic control group. The intervention of metformin sig- Glucose Tolerance nificantly reduced the plasma glucose levels 30, 60, 120, and 180 min after the glucose ingestion, and the area under Intervention with DHM (1.0, 0.5 g/kg BW) or metformin the glucose response curve. (50 mg/kg) did not significantly affect food or water intake The serum insulin level increased significantly in the in db/db mice. As Figures 2 and 3 shown, DHM interven- metabolically abnormal obese db/db mice compared to tion resulted in a dose-related reduction in FBG and that in the normal control group. We observed significant HbA1c. As reported in the literature, metformin signifi- differences in the HOMA insulin resistance (HOMA-IR) cantly lowered the levels of FBG and HbA1c (Figures 2 index between the DHM-treated (1.0 g and 0.5 g/kg BW), and 3) in metabolically abnormal obese db/db mice. In the metformin-treated (50 mg/kg) and untreated metabolically OGTT, the metabolically abnormal obese db/db mice that abnormal obese db/db mice. As Figure 5 shows, DHM or received oral DHM treatment (1.0 and 0.5 g/kg) for 8 metformin intervention led to a significant reduction in the weeks demonstrated a significant suppressed of elevated HOMA-IR index compared with that of the untreated plasma glucose levels 30, 60, 120, and 180 min after the metabolically abnormal obese db/db mice. ingestion of a single high dose of glucose (Figure 4A). Correspondingly, the area under the glucose response Reduced Blood Lipid Levels Regulated curve (AUC, min·mmol/L, Figure 4B) in the DHM-treated Inflammatory Mediators The serum levels of TC and TG increased significantly, Table 2 Effect Of DHM On Liver Weight And Liver Weight/body revealing dyslipidemia in the metabolically abnormal Weight In Mice obese db/db mice. Compared with those of the obese Treatment Dose Liver Liver Weight/Body control mice, serum the concentrations of TC and TG (g/kg) Weight (g) Weight (100%) decreased significantly in the Met and DHM intervention Control – 1.41±0.23** 3.92±0.31** groups (shown in Table 3). – DM 2.99±0.24 4.30±0.34 Table 4 shows the parameters of redox behavior in meta- Met 0.05 2.62±0.35* 3.92±0.25* bolically abnormal obese mice. Compared to the model con- DHM 0.5 2.65±0.31* 3.93±0.31* DHM 1.0 2.61±0.33* 3.97±0.21* trol group, the group treated with DHM for 8 weeks exhibited altered levels of oxidative stress markers, demonstrating the Notes: Different from model control group: *P < 0.05, **P < 0.01. Number of mice= 10. antioxidant effects of DHM. The level of malondialdehyde

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Figure 4 OGTT levels in db/db mice at week 8. Compared to diabetic control mice, metabolically abnormal obese db/db mice (groups 4 and 5) that received oral DHM (1.0 and 0.5 g/kg) intervention for 8 weeks exhibited a signiﬁcant suppression of elevated plasma glucose levels 30, 60, 120, and 180 min after the ingestion of a single high dose of glucose (Figure 4A), as well as a decreased area under the glucose response curve (AUC, min·mmol/L, Figure 4B). Normal: C57BL, DM: untreated metabolically abnormal obese db/db mice, Met: db/db mice treated with metformin, DHM-h, l: db/db mice treated with DHM at 1.0 and 0.5 g/kg BW. *p<0.01 compared with the DM group.

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necrosis factor alpha (TNF-α) and IL-1β (Table 4) compared with those in the metabolically abnormal obese group.

Histological Profiles Of The Effect Of DHM On Lesion Severity In Mice The effects of DHM as determined by histopathologic examination are shown in Figure 6. The pancreas of the normal control group showed no significant histopathological changes and normal islet cell architecture (Figure 6A). The metabolically abnormal obese group exhibited evidence of severe damage characterized by a reduced number and area of islets, the transformation of the borders, vacuolation, and the degranulation of cells fi Figure 5 HOMA-IR index in db/db mice at week 18. DHM treatment signi cantly (Figure 6B). However, DHM intervention restored the reduced the HOMA-IR index. Normal: C57BL, DM: untreated db/db mice, Met: metabolically abnormal obese db/db mice treated with metformin, DHM-h and damaged islets and obviously ameliorated their structure DHM-l: db/db mice treated with DHM at 1.0 and 0.5 g/kg BW. ##p<0.05 compared with the normal group. **p<0.05 compared with the DM group. (Figure 6D). Islet fibrosis, as an important outcome of progressive β-cell failure, plays an important role in the progression (MDA), the end-product of lipid peroxidation, increased and development of type 2 diabetes.38,40 The effects of significantly in the model group versus the normal controls. DHM intervention on the attenuation of fibrosis are shown DHM intervention notably reduced MDA levels. Compared in Figure 7. As demonstrated by Masson trichrome stain- with that in the normal control group, the activity of the ing, islet fibrosis within the islets was significantly antioxidant enzyme superoxide dismutase (SOD) was signif- increased in the diabetic model mice (Figure 7B). icantly lower (P < 0.05) in the metabolically abnormal obese However, it was dramatically reduced in the DHM-treated fi mice. Moreover, DHM treatment yielded signi cantly higher group (Figure 7D), as revealed by the percent area stained activity of antioxidant enzymes. per islet section. It has been reported previously in the literature that The pathological deterioration of the vessels was demon- fl low-grade chronic in ammation play a key role in periph- strated by thicker and rough vessel inner walls, extensive fi eral insulin resistance, which signi cantly increases the arterial plaques containing foam cells, inflammatory cells, 36,37 fl risk of developing T2DM. Controlling in ammation cholesterol crystals, and tissue calcification in the metaboli- may be a core method for improving insulin sensitivity cally abnormal obese models. The pathological changes and subsequently reducing the risk of developing T2DM. decreased visibly in the DHM-treated group, compared with This may also reduce the risk of cardiovascular disease in the model group. The vessel walls were slightly thickened and this high-risk group.2,38,39 Our results indicated that DHM rough, and there were fewer arterial plaques, in the DHM- treatment significantly decreased the serum levels of tumor treated mice.

Effect Of DHM On Insulin Receptor Table 3 Effect Of DHM On Lipid Proﬁle In Mice Substrate 1 (ISR-1) And Phospho-Insulin Treatment Dose (g/kg) TC TG Tyr612 (mmol/L) (mmol/L) Receptor Substrate 1 (p-ISR-1 )

Control – 1.01±0.23** 0.47±0.11** Protein Expression In Liver Tissue DM – 2.39±0.44 1.28±0.23 We examined the protein expression of insulin receptor sub- Met 0.05 2.21±0.35 1.05±0.15* strate-1 and phosphorylated insulin receptor substrate-1 DHM 0.5 2.04±0.31* 1.02±0.13* (p-ISR-1Tyr612, the active form) and other major constituents DHM 1.0 2.01±0.33* 1.09±0.11* of the insulin signaling pathway to elucidate the molecular Notes: Different from model control group: *P < 0.05, **P < 0.01. Number of mechanism of the alleviation of insulin resistance and hyper- mice= 10. 41–43 Abbreviations: TC, serum total cholesterol; TG, triglyceride. glycemia (shown in Figure 8A). As shown in Figure 8B,

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Table 4 Effect Of DHM On Biochemical Parameter In Mice

Treatment Dose (g/kg) SOD (KU/mL) MDA (mmol/L) TNF-α (ng/L) IL-1β (ng/mL)

Control – 3.5 ± 0.2** 2.65 ± 0.13** 10.2 ± 1.1** 0.25 ± 0.03** DM – 0.8 ± 0.3 5.74 ± 0.20 36.0 ± 3.4 0.74 ± 0.12 Met 0.05 2.1 ± 0.3** 3.04 ± 0.21** 36.4 ± 5.5 0.29 ± 0.16** DHM 0.5 2.8 ± 0.4** 3.64 ± 0.17** 23.9 ± 3.3** 0.34 ± 0.10** DHM 1.0 2.3 ± 0.4** 3.07 ± 0.22** 26.8 ± 5.4** 0.34 ± 0.07**

Notes: Different from model control group: **P < 0.01. Number of mice= 10. Abbreviations: IL-1β, interleukin-1β; TNF-α, tumor necrosis factor alpha; MDA, malondialdehyde; SOD, superoxide dismutase. the level of phosphorylation of ISR-1Tyr612 was reduced sig- Discussions nificantly in the liver tissue of db/db mice compared with that Exploring new functional components from traditional Tyr612 of the normal control group. Increased p-ISR-1 level was medicine is a wise strategy.12,13 This paper aimed to eval- observed in the DHM group. The administration of DHM uate the antidiabetic, lipid-lowering and antioxidative Tyr612 induced a higher level of phosphorylation of ISR-1 than function of DHM and to explore the underlying mechan- that of the diabetic model group. There was statistically sig- ism. Pancreatic β-cell failure and apoptosis play a primary Tyr612 nificant between groups. As expected, p-ISR-1 level and pivotal role in the pathophysiologic progression and increased significantly in the Met group. development of T2DM.3,36 We demonstrated that DHM

Figure 6 Histopathological changes in the pancreas of db/db mice. The diabetic control group showed evidence of severe damage characterized by a reduced number and area of islets, the transformation of borders, vacuolation, and the degranulation of cells (B), whereas the normal control group exhibited islets with typical histological structure in the pancreas (A). The administration of Met and DHM restored the damaged islets, obviously ameliorating their structure (C and D). Red arrow symbols mean pancreatic lesions.

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Figure 7 Histopathological changes in islet fibrosis in db/db mice. The diabetic control group showed evidence of severe damage characterized by the degranulation of β- cells and islet fibrosis (B), whereas the normal control group exhibited islets with typical histological structure in the pancreas (A). The administration of Met and DHM prevented islet fibrosis (C and D). has an antidiabetic effect in db/db mice, which was sup- Oxidative stress has been linked to obesity and associated ported by its hypoglycemic and hypolipidemic effects, and cardiovascular diseases. Increased oxidative stress is con- its ability to protect islet cells and delay islet cell fibrosis. sidered a critical cause of insulin resistance, dyslipidemia, More importantly, the present study indicated that DHM β-cell dysfunction, islet fibrosis, and impaired glucose treatment reduced insulin resistance, as evidenced by the tolerance and ultimately leads to T2DM.44 We assessed decrease in the HOMA-IR index. This may partially the levels of inflammatory mediators and oxidative stress explain the mechanism of the hypoglycemic effect. markers (malondialdehyde) and antioxidant enzyme In humans, β-cell mass expansion has been considered a (superoxide dismutase) activity. Oxidative stress has been response to the increased insulin demands imposed by insulin recognized as a multifactorial etiological factor contribut- resistance. Excessive β-cell compensation leads to the dys- ing to insulin resistance and the fibrosis of various organs function and destruction of fibrotic islets which are important in the body. Chronic islet inflammation, fibrosis and the features of pathological deterioration. Meanwhile, islet fibro- loss of parenchymal cells in the pancreas synergistically sis may accelerate β-cell destruction, such as that in chronic contribute to the irreversibility of the progression of the pancreatitis, and induce the disruption of β-cell connectivity, disease. Pancreatic fibrosis severely impairs the exocrine consequently further impairing insulin secretion. and endocrine functions of the pancreas, diminishing the According to the previous literature, islet fibrosis patient’s quality of life. aggravates pathological deterioration in the late stage of These results suggest that DHM may have protective β-cell dysfunction during the progression of T2DM.36,40 effects against chronic inflammation and oxidative stress. In

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Figure 8 Protein expression of ISR-1 and p-ISR-1Tyr612. The protein expression of insulin receptor substrate 1 and phosphorylated insulin receptor substrate 1 in liver tissue are shown in (A). The quantitative analysis is shown in (B). The results indicate that the levels of phosphorylation of ISR-1 Tyr612 were markedly reduced in the db/db mice compared with the normal control group. Increased ISR-1 Tyr612 phosphorylation was observed in the liver tissue of the DHM group. The DHM-administered group showed higher ISR-1 Tyr612 phosphorylation than that in the diabetic model group, and the difference was statistically significant. As expected, ISR-1 Tyr612 phosphorylation increased significantly in the Met group. *p<0.05 compared with the DM group. # p<0.05 compared with the control group. the animal trial, DHM exerted strong hypoglycemic and hope to T2DM treatment and management since therapeutic hypolipidemic effects, reduced insulin resistance, and intervention reduced pancreatic injury, protected islet cells decreased the relative abdominal fat weight. Treatment with and delayed islet cell fibrosis in the animals. DHM attenuated the progression of insulin resistance and For the obesity control effect, the animal weight gain pancreatic fibrosis in fatty db/db mice. This discovery brings coincided with the abdominal fat raise. The lipid

2246 submit your manuscript | www.dovepress.com Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy 2019:12 DovePress Dovepress He et al deposition in the arterial inner wall accelerated the pro- Acknowledgements gression pathological deterioration of aorta. This study The authors gratefully acknowledge the financial assis- showed that natural product DHM might protect the vas- tance of the National Major Drug Discovery Projects cular endothelial function by lowering plasma lipid levels (No: 2017ZX 09301025). The authors thank the scientific and blocking the oxidation of LDL. editors from American Journal Experts for providing pro- Insulin resistance is a set of pathophysiological complica- fessional English language editing of this paper. tions involved in the induction and worsening of many chronic diseases including T2DM. In the T2DM patient, insulin resistance is a pathological state in which the target cells in liver, Disclosure Dr Long Cheng reports a grant from National Major Drug skeletal muscle, and adipose tissue fail to respond properly to Discovery Projects (No: 2017ZX 09301025), during the insulin, resulting in hyperglycemia. Serine/tyrosine phosphor- conduct of the study. The authors report no other conflicts ylation of IRS-proteins has been implicated in the modulation of interest in this work. of insulin signaling and may contribute to the development of insulin resistance.41,42,45–47 This study confirmed that DHM administration elevated IRS-1 Tyr612 protein phosphorylation References in liver tissue when compared with that in the control, suggest- 1. Rayyan Assi H, Ziv A, Dankner R. The metabolic syndrome and its components are differentially associated with chronic diseases in a ing that DHM restored the impaired insulin signaling pathway. high-risk population of 350 000 adults: A cross-sectional study. The results indicated that the phosphorylation of IRS-1 caused Diabetes Metab Res Rev. 2019;35:e3121. doi:10.1002/dmrr.v35.4 by DHM administration was strongly related to the improve- 2. Lent-Schochet D, McLaughlin M, Ramakrishnan N, Jialal I. Exploratory metabolomics of metabolic syndrome: a status report. ment of insulin resistance. World J Diabetes. 2019;10:23–36. doi:10.4239/wjd.v10.i1.23 We suggest that IRS-1 (Y612) tyrosine phosphorylation 3. Daskalopoulos G, Karkanaki A, Piouka A, et al. Excess metabolic and cardiovascular risk is not manifested in all phenotypes of poly- may play a major role in IR in T2DM, and p-IRS-1 protein cystic ovary syndrome: implications for diagnosis and treatment. activation may be a reasonable potential molecular target Curr Vasc Pharmacol. 2015;13:788–800. for attenuating the progression of metabolic syndrome. 4. Mogre V, Salifu ZS, Abedandi R. Prevalence, components and associated demographic and lifestyle factors of the metabolic syndrome in type 2 diabetes mellitus. J Diabetes Metab Disord. 2014;13:80. Conclusions doi:10.1186/2251-6581-13-80 fi 5. Haffner SM. Risk constellations in patients with the metabolic syn- In conclusion, our ndings showed that DHM exerts a drome: epidemiology, diagnosis, and treatment patterns. Am J Med. strong hypoglycemic effect and reduces insulin resistance. 2006;119:S3–S9. doi:10.1016/j.amjmed.2006.01.008 In addition, DHM treatment also markedly decreased the 6. Punthakee Z, Goldenberg R, Katz P. C. Diabetes Canada clinical practice guidelines expert. Can J Diabetes. 2018;42(Suppl 1):S10– relative abdominal fat weight. Western blot analysis S15. doi:10.1016/j.jcjd.2017.10.003 revealed that DHM upregulates the IRS-1 (Y612) tyrosine 7. Engin A. The Definition and prevalence of obesity and metabolic syndrome. Adv Exp Med Biol. 2017;960:1–17. doi:10.1007/978-3- phosphorylation, resulting in intracellular transduction of 319-48382-5_1 the insulin signal, improving insulin resistance. Treatment 8. Kim M, Kim IH, Lim MK, Kim Y, Park B. Increased prevalence of fi with DHM attenuates the progression of insulin resistance metabolic syndrome in adult cancer survivors: asian rst report in community setting. Cancer Epidemiol. 2019;58:130–136. and pancreatic fibrosis in fatty db/db mice. Overall, DHM doi:10.1016/j.canep.2018.12.006 may prove to be a promising approach for preventing meta- 9. Kim JH, Yoo JY, Kim HS. Metabolic syndrome in South Korean patients with chronic obstructive pulmonary disease: a focus on bolic syndrome. gender differences. Asian Nurs Res (Korean Soc Nurs Sci). 2019;13:137–146. doi:10.1016/j.anr.2019.03.002 Abbreviations 10. Misra A, Bhardwaj S. Obesity and the metabolic syndrome in developing countries: focus on South Asians. Nestle Nutr Inst Workshop MetS, metabolic syndrome; DHM, dihydromyricetin; db/db, Ser. 2014;78:133–140. doi:10.1159/000354952 C57BL/KsJ-db/db, T2DM, type 2 diabetes mellitus; Met, 11. Misra A, Soares MJ, Mohan V, et al. Body fat, metabolic syndrome and hyperglycemia in South Asians. J Diabetes Complications. metformin; Hb1Ac, glycated hemoglobin A1c; HOMA-IR, 2018;32:1068–1075. doi:10.1016/j.jdiacomp.2018.08.001 homeostatic model assessment of insulin resistance; IRS-1, 12. Zhang F, Xie JM, Zhang YY, Kong LL, Li SC. What is important insulin receptor substrate-1; TCM, traditional Chinese med- during the selection of Traditional Chinese Medicine (TCM) in a health care reimbursement or insurance system? Critical issues of icine; TC, serum total cholesterol; TG, triglyceride; MDA, assessment from the perspective of TCM Practitioners. Value Health malondialdehyde; SOD, superoxide dismutase; IL-1β, inter- Reg Issues. 2013;2:141–146. doi:10.1016/j.vhri.2013.01.004 β α 13. Xu J, Yang Y. Traditional Chinese medicine in the Chinese health leukin-1 ;TNF- , tumor necrosis factor alpha; HDL-C, high care system. Health Policy. 2009;90:133–139. doi:10.1016/j. density liptein cholesterol. healthpol.2008.09.003

Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy 2019:12 submit your manuscript | www.dovepress.com 2247 DovePress He et al Dovepress

14. Zou L, Zhang Y, Sasaki JE, et al. Wuqinxi Qigong as an Alternative 31. Ye X, Pang Z, Zhu N. Dihydromyricetin attenuates hypertrophic scar Exercise for Improving Risk Factors Associated with Metabolic formation by targeting activin receptor-like kinase 5. Eur J Syndrome: A Meta-Analysis of Randomized Controlled Trials. Int J Pharmacol. 2019;852:58–67. doi:10.1016/j.ejphar.2019.02.039 Environ Res Public Health. 2019;16. 32. Zhang YS, Ning ZX, Yang SZ, Wu H. Antioxidation properties and 15. Patel S. Functional food red yeast rice (RYR) for metabolic syndrome mechanism of action of dihydromyricetin from ampelopsis grosse- amelioration: a review on pros and cons. World J Microbiol dentata. Yao Xue Xue Bao. 2003;38:241–244. Biotechnol. 2016;32:87. doi:10.1007/s11274-016-2035-2 33. Zuo Y, Xu Q, Lu Y, et al. Dihydromyricetin induces apoptosis in a 16. Song HP, Wu SQ, Qi LW, et al. A strategy for screening active lead human choriocarcinoma cell line. Oncol Lett. 2018;16:4229–4234. compounds and functional compound combinations from herbal med- doi:10.3892/ol.2018.9220 icines based on pharmacophore filtering and knockout/knockin chro- 34. Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, matography. J Chromatogr A. 2016;1456:176–186. doi:10.1016/j. Turner RC. Homeostasis model assessment: insulin resistance and chroma.2016.06.009 beta-cell function from fasting plasma glucose and insulin concen- 17. Fan L, Zhao X, Tong Q, et al. Interactions of dihydromyricetin, a trations in man. Diabetologia. 1985;28:412–419. doi:10.1007/ flavonoid from vine tea (Ampelopsis grossedentata) with gut micro- bf00280883 biota. J Food Sci. 2018;83:1444–1453. doi:10.1111/1750-3841.14128 35. Wallace TM, Levy JC, Matthews DR. Use and abuse of HOMA – 18. Wan W, Jiang B, Sun L, Xu L, Xiao P, Peterson JM. Metabolomics modeling. Diabetes Care. 2004;27:1487 1495. doi:10.2337/diacare. reveals that vine tea (Ampelopsis grossedentata) prevents high-fat-diet- 27.6.1487 induced metabolism disorder by improving glucose homeostasis in rats. 36. Alejandro EU, Gregg B, Blandino-Rosano M, Cras-Meneur C, β PLoS One. 2017;12:e0182830. doi:10.1371/journal.pone.0182830 Bernal-Mizrachi E. Natural history of -cell adaptation and failure – 19. Gao Q, Ma R, Chen L, et al. Antioxidant profiling of vine tea in type 2 diabetes. Mol Aspects Med. 2015;42:19 41. doi:10.1016/j. (Ampelopsis grossedentata): off-line coupling heart-cutting HSCCC mam.2014.12.002 with HPLC-DAD-QTOF-MS/MS. Food Chem. 2017;225:55–61. 37. Goldenberg R, Punthakee Z. C. Canadian Diabetes Association clin- – doi:10.1016/j.foodchem.2016.11.122 ical practice guidelines expert. Can J Diabetes. 2013;37(Suppl 1):S8 20. Wu J, Wang C, Huang G, et al. Biotransformation of vine tea S11. doi:10.1016/j.jcjd.2013.01.011 (Ampelopsis grossedentata) by solid-state fermentation using medic- 38. Petrie JR, Guzik TJ, Touyz RM. Diabetes, hypertension, and cardiovascular disease: clinical insights and vascular mechanisms. Can J inal fungus Poria cocos. J Food Sci Technol. 2016;53:3225–3232. Cardiol. 2018;34:575–584. doi:10.1016/j.cjca.2017.12.005 doi:10.1007/s13197-016-2297-6 39. Katakami N. Mechanism of Development of Atherosclerosis and 21. Ye L, Wang H, Duncan SE, Eigel WN, O’Keefe SF. Antioxidant Cardiovascular Disease in Diabetes Mellitus.. J Atheroscler activities of vine tea (Ampelopsis grossedentata) extract and its major Thromb. 2018;25:27–39. doi:10.5551/jat.RV17014 component dihydromyricetin in soybean oil and cooked ground beef. 40. Nelson L, Turkel S, Shulman I, Gabbe S. Pancreatic-islet fibrosis in Food Chem. 2015;172:416–422. doi:10.1016/j.foodchem.2014.09.090 young infants of diabetic mothers. Lancet. 1977;2:362–363. 22. Chen T, Zhu S, Lu Y, et al. Probing the interaction of anti-cancer doi:10.1016/s0140-6736(77)91531-8 agent dihydromyricetin with human serum albumin: a typical method 41. Alipourfard I, Datukishvili N, Mikeladze D. TNF-α downregulation study. Anticancer Agents Med Chem. 2012;12:919–928. modifies Insulin Receptor Substrate 1 (IRS-1) in metabolic signaling 23. Dong S, Ji J, Hu L, Wang H. Dihydromyricetin alleviates acetami- of diabetic insulin-resistant hepatocytes. Mediators Inflamm. nophen-induced liver injury via the regulation of transformation, lipid 2019;2019:1–6. doi:10.1155/2019/3560819 homeostasis, cell death and regeneration. Life Sci. 2019. 42. Rashidi B, Azizy L, Najmeddin F, Azizi E. Prevalence of the insulin 24. Huang H, Hu M, Zhao R, Li P, Li M. Dihydromyricetin suppresses receptor substrate-1(IRS-1) Gly972Arg and the insulin receptor sub- the proliferation of hepatocellular carcinoma cells by inducing G2/M strate-2(IRS-2) Gly1057Asp polymorphisms in PCOS patients and arrest through the Chk1/Chk2/Cdc25C pathway. Oncol Rep. non-diabetic healthy women. J Assist Reprod Genet. 2012;29:195– – 2013;30:2467 2475. doi:10.3892/or.2013.2705 201. doi:10.1007/s10815-011-9693-7 25. Li H, Yu F, Sun X, Xu L, Miu J, Xiao P. Dihydromyricetin amelio- 43. Burgos-Ramos E, Gonzalez-Rodriguez A, Canelles S, et al. rates memory impairment induced by acute sleep deprivation. Eur J Differential insulin receptor substrate-1 (IRS1)-related modulation – Pharmacol. 2019;853:220 228. doi:10.1016/j.ejphar.2019.03.014 of neuropeptide Y and proopiomelanocortin expression in nondiabetic 26. Li Q, Wang J, Zhu X, et al. Dihydromyricetin prevents monocrota- and diabetic IRS2-/- mice. Endocrinology. 2012;153:1129–1140. line-induced pulmonary arterial hypertension in rats. Biomed doi:10.1210/en.2011-1278 – Pharmacother. 2017;96:825 833. doi:10.1016/j.biopha.2017.10.007 44. Tangvarasittichai S. Oxidative stress, insulin resistance, dyslipidemia 27. Peng J, Zhang J, Zhang L, Tian Y, Li Y, Qiao L. Dihydromyricetin and type 2 diabetes mellitus. World J Diabetes. 2015;6:456–480. improves vascular hyporesponsiveness in experimental sepsis via doi:10.4239/wjd.v6.i3.456 attenuating the over-excited MaxiK and KATP channels. Pharm 45. Evert M, Sun J, Pichler S, Slavova N, Schneider-Stock R, – Biol. 2018;56:344 350. doi:10.1080/13880209.2018.1478430 Dombrowski F. Insulin receptor, insulin receptor substrate-1, Raf-1, 28. Ren Z, Yan P, Zhu L, et al. Dihydromyricetin exerts a rapid antidepres- and Mek-1 during hormonal hepatocarcinogenesis by intrahepatic sant-like effect in association with enhancement of BDNF expression pancreatic islet transplantation in diabetic rats. Cancer Res. fl and inhibition of neuroin ammation. Psychopharmacology. 2004;64:8093–8100. doi:10.1158/0008-5472.CAN-04-2040 2018;235:233–244. doi:10.1007/s00213-017-4761-z 46. Fernandez-Twinn DS, Alfaradhi MZ, Martin-Gronert MS, et al. 29. Shen Y, Lindemeyer AK, Gonzalez C, et al. Dihydromyricetin as a Downregulation of IRS-1 in adipose tissue of offspring of obese mice novel anti-alcohol intoxication medication. J Neurosci. 2012;32:390– is programmed cell-autonomously through post-transcriptional mechan- 401. doi:10.1523/JNEUROSCI.4639-11.2012 isms. Mol Metab. 2014;3:325–333. doi:10.1016/j.molmet.2014.01. 30. Wang YC, Liu QX, Zheng Q, et al. Dihydromyricetin Alleviates 007 Sepsis-Induced Acute Lung Injury through Inhibiting NLRP3 47. Badin PM, Louche K, Mairal A, et al. Altered skeletal muscle lipase Inflammasome-Dependent Pyroptosis in Mice Model. Inflammation. expression and activity contribute to insulin resistance in humans. 2019. Diabetes. 2011;60:1734–1742. doi:10.2337/db10-1364

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